Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-12-15
2001-10-09
LeGuyader, John L. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S325000, C435S455000, C435S458000, C424S450000
Reexamination Certificate
active
06300321
ABSTRACT:
This application is a US National Stage application of co-pending PCT application PCT/FR98/01041, filed May 25, 1998. This application also claims the benefit of foreign priority under 35 U.S.C. § 119(a) to application FR97/06549, filed May 28, 1997 in France.
The present invention relates to new compounds for transferring nucleic acids into cells. These new compounds are more particularly related to the lipopolyamine family and contain amidine functional groups. These compounds can be used for transferring nucleic acids of interest into various cell types, both in vitro and in vivo or ex vivo.
With the development of biotechnology, the possibility of effectively transferring nucleic acids into cells has become a necessity. It may involve the transfer of nucleic acids into cells in vitro, for example, for the production of recombinant proteins, or in the laboratory for studying the regulation of the expression of genes, the cloning of genes, or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the creation of transgenic animals, the production of vaccines, labelling studies or also therapeutic approaches. It may also be the transfer of nucleic acids into cells ex vivo, in approaches including bone marrow transplants, immunotherapy or other methods involving the transfer of genes into cells collected from an organism for the purpose of their subsequent readministration.
Various types of synthetic vector have been developed to improve the transfer of nucleic acids into cells. Among these vectors, cationic lipids possess advantageous properties. These vectors consist of a cationic polar part which interacts with the nucleic acids, and a hydrophobic lipid part which promotes cellular penetration. Specific examples of cationic lipids are in particular the monocationic lipids (DOTMA: Lipofectin®); some cationic detergents (DDAB); lipopolyamines, and in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES), whose preparation has been described, for example, in Patent Application EP 394 111. Another lipopolyamine family is represented by the compounds described in Patent Application WO 97/18185. incorporated into the present by way of reference.
The present invention relates to a new type of agent for transferring nucleic acids, possessing particularly advantageous properties. More precisely, the compounds according to the present invention are cationic lipids carrying a novel cationic region, conferring improved properties on the molecules. This cationic part is represented more precisely by a particular polyamine, carrying one or more amidine functional groups.
A first subject of the invention relates more precisely to compounds in D, L or DL form, as well as its salts, of general formula (I):
in which:
R
1
, R
2
and R
3
represent, independently of each other, a hydrogen atom or a group —(CH
2
)
q
—NRR′ with
q being an integer from 1 to 6 inclusive, the values of q being independent of each other between the different groups R
1
, R
2
and R
3
, and
R and R′ represent, independently of each other, a hydrogen atom or a group of formula (II):
in which r is an integer which may vary from 0 to 6 inclusive, and the R
5
groups represent, independently of each other, a hydrogen atom or a hydrocarbon residue, it being understood that at least one of the groups R
1
, R
2
and R
3
contains at least one group of formula (II),
m and n reptesent, independently of each other, an integer which may vary from 1 to 6 inclusive with, when n is greater than 1, m being capable of having different values and R
3
different meanings within the general formula (I),
p represents an integer which may vary from 1 to 6 inclusive, and
R
4
represents a group of general formula (III):
in which:
R
7
and R
8
represent, independently of each other, a hydrogen atom or a lipophilic group, at least one of the groups R
7
and R
8
being different from hydrogen,
t is an integer chosen from 0 to 10 inclusive with R
6
, X, Y and s being capable of having different meanings within the different units [X—(CHR
6
)
s
—Y] when t is an integer greater than 1,
X represents an oxygen or sulphur atom or an amino or alkylamino group, the alkyl substituent being linear or branched and containing 1 to 8 carbon atoms,
Y represents a carbonyl group or a methylene group,
R
6
represents a hydrogen atom or a natural amino acid side chain, substituted where appropriate and
s represents an integer varying between 1 and 10 inclusive with, when s is equal to 1, R
6
representing a natural amino acid side chain, substituted where appropriate, and when s is greater than 1, R
6
representing a hydrogen atom.
The term “DL form” as used above means a mixture of D and L forms in any proportion, and for example in equal proportions.
For the purposes of the invention, “hydrocarbon residue” is understood to mean any aliphatic or aromatic alkyl, carbamate or acyl substituent which is optionally halogenated. Among the aliphatic residues, there may be mentioned more particularly cyclic or noncyclic, linear or branched, saturated or unsaturated aliphatic residues which are optionally halogenated. More preferably, it is an aliphatic residue containing 1 to 10 carbon atoms. In particular, it represents alkanoyl, alkyl, and alkylcarbamate substituents such as, for example, formyl, butyl, tert-butyl or tert-butylcarbamate substituents. According to a variant of the invention, R
5
represents tert-butylcarbamate.
Among the aromatic residues, there may be mentioned more particularly benzyl and its substituted derivatives such as chlorobenzyl, benzylcarbamate or chlorobenzylcarbamate.
Preferably, R
5
represents tert-butylcarbamate, benzylcarbamate or chlorobenzylcarbamate.
In a first variant of the invention, one of the R
5
groups represents a hydrogen atom and the other an aliphatic residue containing 1 to 10 carbon atoms.
In a second variant of the invention, one of the R
5
groups represents a hydrogen atom and the other an aromatic residue preferably chosen from benzyl and its derivatives.
In another variant of the invention, the two R
5
groups represent hydrogen atoms.
Advantageously, when R
1
, R
2
and/or R
3
are different from hydrogen and comprise the general formula (II), q takes the value 2 or 3 and r is equal to 0.
Preferably, in the general formula (I), m is chosen from 2, 3 and 4.
As indicated above, in formula (III), at least one of the groups R
7
and R
8
represents a lipophilic group. For the purposes of the invention, “lipophilic group” is understood to mean any group of the hydrophobic, lipid type which promotes cellular penetration known to persons skilled in the art. It may represent in particular one or more aliphatic fatty chains, a steroid derivative, a natural or synthetic lipid, preferably capable of forming lamellar or hexagonal phases, or optionally a combination of these. It may represent more particularly a linear or branched, saturated or unsaturated aliphatic radical containing 5 to 22 carbon atoms which is optionally 7 halogenated. It may also represent a steroid derivative or a group (CH
2
)
u
—NH—R
9
in which u is an integer between 2 and 6 inclusive and R
9
is an acyl radical such as for example cholesteryl formate, arachidonyl or cholic acid.
Other examples of steroid derivatives are in particular cholesterol, cholestanol, 3-&agr;-5-cyclo-5-&agr;-cholestan-6-&bgr;-ol, cholic acid, cholesteryl formate, chotestanyl formate, 3&agr;,5-cyclo-5&agr;-cholestan-6&bgr;-yl formate, cholesterylamine, 6-(1,5-dimethylhexyl)-3a,5a-dimethylhexadecahydrocyclopenta[a]cyclopropa[2,3]cyclopenta[1,2-f]naphthalen-10-ylamine, or cholestanylamine.
Preferably, the lipophilic group is an aliphatic radical containing between 10 and 22 carbon atoms, preferably 14, 16, 17, 18 or 19 carbon atoms. In particular, the lipophilic groups (CH
2
)
13
CH
3
(CH
2
)
15
CH
3
, (CH
2
)
16
CH
3
, (CH
2
)
17
CH
3
, (CH
2
)
18
CH
3
and oleyl may be
Byk Gerardo
Dubertret Catherine
Scherman Daniel
Aventis Pharma S.A.
Epps Janet
Krupen Karen I.
LeGuyader John L.
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